The modification of polymeric matrices by adding calcium-phosphate derivatives has been proven an effective strategy for tailoring the properties of scaffolds employed in bone tissue engineering. In this regard and, considering the biomechanics of bone as well as the durotactic response of osteoblasts, this study builds on the hypothesis that the preparation of novel Gellan Gum (GG)-Hydroxyapatite (HA) hydrogel composites could benefit the mechanical profile of matrices as well as the cell-substrate interaction in favor of cell recruitment and growth.
To this purpose, HA microparticles at different concentrations (10 and 20%) were successfully incorporated into GG based hydrogels. The composites were characterized by Scanning Electron Microscopy (SEM) coupled with Energy Dispersive Spectroscopy (EDS), Fourier Transformed Infrared Spectroscopy (FTIR), X-Ray Diffraction Analysis (XRD), Micro Computed Tomography (μ-CT) Analysis and Thermogravimetric Analysis (TGA). In vitro degradation and swelling studies were conducted in PBS solution, while the GG/HA composites were subjected to Dynamic Mechanical Analysis (DMA). The efficacy of the GG/HA matrices to precipitate apatite in Simulated Body Fluid (SBF) was evaluated, while the cell-matrix interactions were studied by seeding the composites with human osteoblast-like cells. The cell-viability was assayed by staining the cell-loaded composites with calcein-AM, Texas Red-Phalloidin and DAPI-blue and observing by confocal microscope.
The image (SEM, μ-CT)-assisted microstructural characterization of the GG/HA composites reveals hydrogels with high porosity (>80%) and average pore size of 260 μm. GG/HA composites demonstrate high water retention ability during swelling studies, while the weight loss did not exceed 8% during the degradation studies. FTIR and XRD detected peaks typical of hydroxyapatite. The thermal curves of the composites disclose an initial weight loss due to moisture removal and two subsequent degradation phases due to the polymeric component decomposition. The matrices exhibit increasing storage modulus (E’) and decreasing loss factor (tan δ) as a function of frequency during DMA analysis, while composites containing 20% HA possess always higher E’ and lower tan δ values. GG/HA composites develop bioactivity in the form of multiple agglomerates of apatite crystals since the 3rd post-immersion day in SBF. Viability assays indicated that the human osteoblast-like cells seeded on the GG/HA composites were metabolically active.
Concluding, the modification of GG-based hydrogels by HA in different concentration appears to result in composites that meet several scaffold-design criteria and to allow the tailoring of their mechanical performance. However, further optimization is required in order to improve cell adhesion and to promote cell growth.